Protein phosphatase-1 activation and association with the retinoblastoma protein in colcemid-induced apoptosis.

Protein phosphatase-1 (PP1) is cell cycle regulated and potentially related to apoptosis. We studied PP1 in HeLa cells exposed to colcemid, which leads first to mitotic block, then to cell death within 72 h. The soluble PP1 activity, which was low at 14 h (mitosis), was then reversibly activated (maximally around 48 h), with parallel changes in the protein levels of the alpha, gamma1 and delta PP1 isoforms. PP1 activation suggested its involvement in dephosphorylating proteins relevant to apoptosis. Among these, we examined the retinoblastoma protein (pRb). This was found hyperphosphorylated at 14 h. Hypophosphorylated pRb appeared at 24 h, increased at 48 h, and was the only form left at 72 h. PP1 was found to associate with immunoprecipitated pRb, as indicated by PP1 activity assays on the pRb-immunocomplexes. The pRb-associated PP1 activity was low at 14 h, maximal at 24 h, low again by 72 h and was due to PP1delta. The presence of active PP1 suggests its involvement in pRb dephosphorylation.

Protein tyrosine phosphatase PTP-S binds to the juxtamembrane region of the hepatocyte growth factor receptor Met.

We reported previously that a protein tyrosine phosphatase (PTP) activity is associated with the immunoprecipitated hepatocyte growth factor (HGF) receptor, also known as Met. The activity increased reversibly when Met was stimulated by HGF and decreased when Met was inactivated by PMA. To identify the PTP-binding region, we used deletion mutants of the receptor beta-subunit. The PTP activity did not associate with Tpr-Met, a construct containing residues 1010-1390 of Met fused to Tpr. In contrast, PTP activity was present when the expressed protein contained the full juxtamembrane region (residues 956-1390 of Met) or part of this region (residues 957-1390 or 995-1390), indicating that the PTP-binding region is between residues 995 and 1009. This region includes Tyr1003, a site involved in Met downstream signalling. Incubation of Met immunoprecipitated from GTL-16 cells with an 8-mer phosphopeptide derived from residues 1003-1010 induced a marked decrease in the associated PTP activity, suggesting that the peptide reproduced the PTP-binding region. Mutation of Glu, Asp or Arg at positions -4, -1 or +1 respectively relative to Tyr1003 in a 9-mer peptide (residues 999-1007) abolished the ability of the peptide to decrease the PTP activity associated with Met. Phosphorylation of Tyr1003 was not required for PTP binding, since: (1) both phospho- and dephospho-peptides on a solid bead bound PTP activity from a GTL-16 cell extract, and (2) PTP activity was associated with a Met deletion mutant lacking residues 1-955 in which Tyr1003 had been changed into Phe. In order to partially purify the PTP from the GTL-16 cell extract, an affinity column was prepared using the Met-derived peptide comprising residues 998-1007. Less than 0.1% of the total cellular PTP was retained by the column, and was eluted with low salt concentrations. Using antibodies, this PTP was identified as PTP-S, a soluble PTP present in epithelial cells and fibroblasts.

Association of protein phosphatase-1delta with the retinoblastoma protein and reversible phosphatase activation in mitotic HeLa cells and in cells released from mitosis.

The retinoblastoma gene product (pRb) is dephosphorylated at the exit from mitosis and protein phosphatase-1 (PP1) seems to be responsible for such dephosphorylation. Three isoforms of PP1 exist in mammalian cells, alpha, gamma1 and delta, with differential subcellular localization and potentially different targeting subunits and functions. In order to identify which isoform dephosphorylates pRb, we used isoform-specific antibodies and analyzed the association of the PP1 isoforms with pRb in nocodazole-blocked (mitotic) HeLa cells and in cells released from the mitotic block (early G1). PP1delta was found associated with the pRb immunoprecipitated from a mitotic cell extract, whereas neither PP1gamma1 nor PP1alpha were detected. In G1 cells progressively less pRb and of lower Mr was detected in anti-PP1delta immunocomplexes, and pRb had almost disappeared by 8 h. The PP1 associated with pRb was inactive at mitosis, but underwent a quick activation as cells exited from mitosis, with a peak at 1 h. Then the activity decreased progressively and disappeared by 8 h. [32P]labeled pRb, obtained from G2 cells, was dephosphorylated "in vitro" by PP1delta obtained from early G1 cells. Altogether, the results indicated that PP1delta associated with pRb and may be responsible for the phosphatase activity detected in the pRb complexes, supporting the hypothesis that PP1delta may be the isoform that dephosphorylates pRb.

Phosphorylation of protein phosphatase-1 isoforms by cdc2-cyclin B in vitro.

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Villa-Moruzzi, FEBS Lett 304: 211-215, 1992). In the present study we show that all the three Phosphatase-1 isoforms, alpha, gamma 1, delta, are phosphorylated by cdc2-cyclin B. Phosphorylation is specific for this kinase and involves a C-terminal Thr. This site is most likely Thr 320 in alpha (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in gamma 1, delta and in the testis-specific gamma 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-1 purified from skeletal muscle, which is a mixture of the alpha, gamma 1 and delta isoforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphopeptide map. This is in agreement with the different sequences of the isoforms in the C-terminal regions and may be useful to distinguish the isoforms in extracts from metabolically-labelled cells. Our results suggest that all the Phosphatase-1 isoforms may be potentially regulated at M-phase.

Protein phosphatase-1 alpha, gamma 1, and delta: changes in phosphorylation and activity in mitotic HeLa cells and in cells released from the mitotic block.

Protein phosphatase-1 is phosphorylated "in vitro" by cdc2-cyclin B (E. Villa-Moruzzi, FEBS Lett. 304, 211-215, 1992). In the present study the phosphatase-1 isoforms alpha, gamma 1, and delta were analyzed in mitotic (nocodazole-blocked) HeLa cells. Phosphorylation on threonine increased in gamma 1 and delta at mitosis. alpha was phosphorylated only in mitotic cells and mainly on serine. Exposure of permeabilized mitotic cells to a peptide that inhibits cdc2 decreased the phosphorylation of the isoforms. Cell fractionation indicated that phosphatase-1 was over 90% inactivated and phosphorylated in the soluble, but not in the chromosomal fraction of mitotic cells. Immunoprecipitation from the mitotic soluble fraction indicated that only gamma 1 and delta, but not alpha, were inactivated. Altogether the data pointed to a correlation between phosphatase-1 inactivation and phosphorylation in mitotic cells. cdc2-cyclin B might be the kinase (or one of the kinases) that phosphorylates phosphatase-1. In cells released from the mitotic block, the phosphatase-1 activity in the soluble, but not in the nuclear fraction, increased progressively, reaching control values by 16 h. Immunoprecipitation indicated that the increase in activity was due to alpha and delta only. On the other hand, the activity of gamma 1 remained low, and this was also the only isoform that remained phosphorylated, though less than in mitotic cells. Also in the case of the cells released from mitosis, a correlation may exist between phosphorylation and inactivation of phosphatase-1. However, the regulation of phosphatase-1 is complex and may involve also regulatory subunits that are still unknown. Altogether, the results indicated the differential regulation of the phosphatase-1 isoforms both at mitosis and in G1 cells.

Phosphorylation of phosphatase-1alpha in cells expressing v-src.

Phosphatase-1 (PP1) is phosphorylated "in vitro" by the tyrosine-kinases c-src, v-src and v-abl. In the case of src, this induces enzyme inactivation. We investigated whether in NIH-3T3 cells expressing v-src (A4 cells) PP1 was phosphorylated on Tyr and inactivated. In mammalian cells, three PP1 isoforms are present: PP1alpha, PP1gamma1 and PP1delta. In A4 cells the three PP1 isoforms were all phosphorylated on Ser, but only PP1alpha was also phosphorylated on Tyr. A lower level of PP1 phosphorylation, and on Ser only, was found also in wild-type NIH-3T3 cells. In A4 cells most of Tyr-phosphorylated PP1alpha was cytosolic. Also the PP1 activity was decreased in the cytosol of the A4 cells. Assay of the three immunoprecipitated PP1 isoforms indicated that only PP1alpha was inactivated. Altogether the data suggest that PP1alpha might be a target of v-src "in vivo".

Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice.

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1.

Phosphorylation of the inhibitor-2 of protein phosphatase-1 by cdc2-cyclin B and GSK3.

cdc2-cyclin B activates protein phosphatase-1 (PP1) "in vitro", phosphorylates both catalytic subunit and inhibitor-2 (I2) and both processes are inhibited by a cdc2-inhibitory peptide. We compared the phosphorylation of I2 by cdc2-cyclin B and by the PP1-activator Glycogen Synthase Kinase 3 (GSK3). Each kinase introduced less than 0.1 mol phosphate/mol into I2 bound to PP1 and the same two tryptic phosphopeptides were obtained from I2, which contained phospho-T only. The same results were obtained also with isolated I2 phosphorylated by GSK3. Since GSK3 phosphorylates only T-72, cdc2-cyclin B is also likely to phosphorylate this site. This was confirmed by using I2 that had been mutated at this site. On the other hand cdc2-cyclin B introduced up to 0.8 mol/mol phosphate into isolated I2 and four phosphopeptides were obtained. The two new peptides contained phospho-T and one of them also phospho-S. These data indicate the presence of at least one T and one S that are phosphorylated only by cdc2-cyclin B and are accessible on isolated I2 only.